Process for preparing beta-fructofuranosidase enzyme and a process for producing fructooligosaccharides

ABSTRACT

A process for preparing beta-fructofuranosidase enzyme and a process for producing fructooligosaccharides, in which the preparation of the enzyme is obtained by cultivating the fungus  Aspergillus niger,  either wild or mutated, in a preferably semi-solid culture medium, in order to produce an extracellular enzyme, which is submitted to transfructosylation for producing fructooligosaccharides comprising sugars which are formed by one unit of sacrose and by two, three and four units of fructose.

FIELD OF THE INVENTION

[0001] The present invention refers to a process for preparingbeta-fructofuranosidase enzyme obtained from the culture of fungi of theAspergillus niger species, of both the wild and mutated types, and itsuse in the production of fructooligosaccharides.

BACKGROUND OF THE INVENTION

[0002] Fructooligosaccharides are sugars found in nature, which, whenconsumed, provide several benefits to the health of a person.

[0003] GF2 (1-kestose), GF3 (nystose) and GF4 (fructofuranosil nystose)are composed of glucose units, to which are bound one, two or even fourfructose units.

[0004] These sugars can be found in a large quantity of foods providedby nature, such as asparagus, banana, garlic, onion, tomato or wheat.Besides giving a sweetish flavor to foods, they are neither cariogenicnor caloric and promote the population growth of the bifidus bacteria inthe intestine, which reduce the activity of the putrefactive bacteria,thereby reducing the development of toxic products by fermentation.

[0005] In view of the benefits described above, the interest forfructooligosaccharides have raised progressively. Consequently,intensive investigations are now being undertaken with the aim ofobtaining these fructooligosaccharides from enzymes. It is known thatthe beta-fructofuranosidase enzyme, which is used in the production offructooligosaccharides by transfer activity can be obtained in differentways, particularly from the cultures of fungi of different species, suchas Aspergillus, Pennicillium, Fusarium, Gloesporium, from the culturesof yeasts, such as Saccharomyces, Rhodotorulla, Pichia, Hansenula,Candida and Aureobasidium, and also from some plants, such as asparagus.It is also known that this enzyme may be prepared in different ways andunder different process conditions. Said enzyme is also known forpromoting the catalyzation of the transfructosylation reaction, which isresponsible for transferring the fructosyl group from a donor to areceptor, which may be sucrose or a fructooligosaccharide, such askestose, nystose, etc. Nevertheless, the structure of this enzyme isstill unknown.

[0006] The manner by which the transfructosylation of the Aspergillusorizac occurs has been studied before, regarding its performance in theformation of fructose oligomers, by using sucrose as raw material.

[0007] It has been observed that the hydrolysis of sucrose with extractsof Pennicillium spinulosum was initially fast and followed by theformation of non-reducing fructooligosaccharides. The hydrolysis ofthese fructooligosaccharides occurred by transferring the fructose unitsto the water and eventually resulted in the complete hydrolysis of thesucrose. The transfructosylation and inversion activities occurred fromthe use of the same enzyme, i.e., the beta-fructofuranosidase (Bealing &Bacon, 19953; Bealing 1953). From U.S. Pat. No. 4,849,356fructooligosaccharides were produced with mycelium extracts, byculturing the fungus Aspergillus phoenics in an adequate culture medium.According to this document, the enzyme is preferably prepared on a solidsubstrate mostly containing sucrose. The beta-fructofuranosidase enzymethus obtained is cell-bound and requires, to be recovered, complexoperations for separating the mycelium from the liquid phase. In thisprior art process, in order to obtain a good yield, the presence ofsucrose is still required in the culture medium. Moreover, the enzymeobtained as described above provides only the formation of lowerfructose oligomers, such as GF2, GF3, with the production of GF4 notbeing demonstrated.

[0008] The formation of fructooligosaccharides has also beeninvestigated, by using cell suspension of several other fungi. Amongthese fungi, the Aspergillus niger ATCC 20611 produced the highest levelof the activity of the beta-fructofuranosidase enzyme, as compared tothe activity of hydrolysis (Hidaka et al, 1988). The enzyme wassubsequentially purified and then characterized.

DISCLOSURE OF THE INVENTION

[0009] In view of the results of these studies, which demonstrated acertain complexity regarding the preparation of thebeta-fructofuranosidase enzyme, it is an object of the present inventionto provide a process for preparing the beta-fructofuranosidase enzyme,which permits a yield in the range from 57% to 60% to be obtained.

[0010] It is also an object of the present invention to provide aprocess which is easy to carry out and of low cost, by using the enzymein the free form. It is still an object of the present invention toproduce fructooligosaccharides, including not only GF2, GF3, but alsoGF4.

[0011] These and other objectives are attained by the provision of aprocess for preparing beta-fructofuranosidase enzyme, comprising thesteps of:

[0012] a) inoculating the spores of the fungus Aspergillus niger in anadequate liquid or semi-solid culture medium;

[0013] b) cultivating the already inoculated fungus, in order to promoteits growth with the formation of mycelium and the production of thebeta-fructofuranosidase enzyme; and

[0014] c) separating the beta-fructofuranosidase enzyme from themycelium and from the culture medium.

[0015] For this purpose, the authors of the present invention haveresearched several types of fungi, which were isolated from soils ofsugar cane regions of Brazil, aiming to obtain much better results inrelation to the enzymatic activity of said fungi in a culture medium,and have found out that a new strain of the fungus Aspergillus nigerproduced higher enzymatic activity in a culture medium.

[0016] This new fungus, which is called Aspergillus niger 489, has beenselected from a bank of microorganisms with more than 2,000 differentstrains of the Biochemistry Laboratory of FEA UNICAMP (Food EngineeringSchool of the University of Campinas, Sao Paulo, Brazil), for it showedhigh efficiency in the production of the beta-fructofuranosidase enzymeand the consequent transfer of sucrose in a fructooligosaccharide (about60% of conversion), and it was submitted to a genetic mutation processwith the drug N-nitrous-nitroguanidine and ultraviolet radiation forproductivity increase.

[0017] The preparation of the enzyme may consist of an extract with nocells or with broken cells, or a purified extract of dry andconcentrated cells. The enzyme may also be purified and concentratedfrom cells and culture medium, in which case there may or may not beused specific systems for breaking the cells when the use of theintracellular fraction is desired.

[0018] The preparation of the present beta-fructofuranosidase may beachieved in a liquid culture medium, which preferably contains 5% ofsucrose, 1% of yeast extract, 1% of peptone and 0.3% of sodium chloride.Nevertheless, such procedure causes the production of intracellularenzyme in relation to the mycelium. Thus, although the Aspergillus niger489 allows a high yield to be obtained in the production of thebeta-fructofuranosidase enzyme, its separation process is industriallycomplex, requiring a certain number of operations for the finalextraction of the enzyme.

[0019] For preparing the enzyme on a semi-solid substrate, all is neededis the presence of a cereal bran, preferably wheat bran, in the culturemedium, the substrate being incubated at a temperature in the range of25 to 40° C., during a period of about four to ten days.

[0020] Although the wheat bran has been mentioned as the preferredmaterial for the formation of the semi-solid culture medium, it isimportant to point out that other brans from cereals containing starch,fibres and proteins may be used together with mineral salts andhumidity.

[0021] It has been verified as adequate a culture medium in which itssolid phase is formed of about 98% of cereal bran, preferably wheatbran, and 2% of mineral salts, such as magnesium sulfate, calciumchloride and potassium phosphate, the semi-solid culture mediumconsisting of about 40% of said solid phase and water as the remainingpart.

[0022] The enzyme obtained according to the above cited procedure is notcell-bound, being in the free form and therefore determining thepossibility of working with the immobilized beta-fructofuranosidase. Theenzyme thus obtained can be easily separated.

[0023] It is important to point out, however, that the fungusAspergillus niger may be cultivated on any other substrate, such as forexample in a solid culture medium and in various other forms, such ascontaining or not containing sucrose in the culture medium. Forinstance, for the preparation of the enzyme in a solid medium, thefungus is pre-cultivated on a substrate composed of a P.D.A. (Potato,Dextrose, Agar) medium, which is sterilized for 20 minutes at 110° C.during four days. It should be noted, however, that the most economicalprocedure is the one which employs the semi-solid culture medium.

[0024] In order to obtain good results, the presentbeta-fructofuranosidase can be used in the production offrutooligosaccharides consisting of one unit of glucose and two, threeand four units of fructose, with a a good development in a pH rangingfrom 5.0 to 9.5. The beta-fructofuranosidase activity reaches itsmaximum values in a pH ranging from 6.0 to 8.5. According to the U.S.Pat. No. 4,317,880, in the transfructolyation process which employs thebeta-fructofuranosidase enzyme recovered from the culture mediumcontaining Pullularia pullulans, the pH must be between 6.3 and 6.7.Yeasts cultures are usually more difficult to be processed, besidesbeing more difficult to be filtered.

[0025] The thermal stability of the enzyme preparations derived fromyeasts is also lower. For example, at high temperatures, the activity ofthis type of enzyme preparation decreases more rapidly than the activityof the enzyme preparations derived from fungi. Thus, the utilization ofenzymes derived from fungi, more specifically the preparations of thepresent beta-fructofuranosidase enzyme, which derives from cultures ofthe fungus Aspergillus niger, are more adequate at higher temperatures.

[0026] As mentioned hereinbefore, the present beta-fructofuranosidasediffers from other beta-fructofuranosidases derived from fungi also forpresenting a better thermal stability. This means that, at risingtemperatures, the activity of the present enzyme is diminished, ascompared to other beta-fructofuranosidase enzymes. High temperatures areoptimum for the preparation of fructooligosaccharides, with the optimumtemperatures for preparing the fructosylation with the presentbeta-fructofuranosidase ranging between 40° C. and 65° C. Therefore,with the adequate choice for the process conditions, which can be easilydetermined, the transfructosylation can be carried out, withoutoccurring any infection of the reaction mixture.

[0027] The immobilization of the present beta-fructofuranosidase may bedesirable, even though partially, for several other purposes, such as,for example, in the utilization in a fixed or fluidized bed. As alreadydescribed, the immobilization of the enzyme can be obtained by employingthe semi-solid substrate containing wheat bran or a bran from any othercereal.

[0028] Another variable characteristic of the presentbeta-fructofuranosidase is that not only GF2 (kestose) and GF3 (nystose)are formed, but also GF4 (fructofuranosil nystose) during the reaction.Due to the low invertase activity of the present enzyme, the inversionof sugars continues to be very low, resulting in the formation of acertain amount of glucose.

[0029] The concentration of sucrose also exhibits a wide range ofvariation, from 25% to 80% (m/m), in which the enzyme activity occurs,with the highest activity values occurring in the range of 40% to 70%(m/m) of sucrose concentration. The sucrose used may be in the pure formor that contained in intermediate molasses of the sugar refining processand the reaction mixtures, which are obtained in the transfructosylationof sucrose, can be analyzed by high pressure liquid chromatography(HPLC).

1. A process for preparing beta-fructofuranosidase enzyme, characterizedin that it comprises the steps of: a) inoculating the spores of thefungus Aspergillus niger in an adequate liquid or semi-solid culturemedium; b) cultivating the already inoculated fungus, in order topromote its growth with the formation of mycelium and the production ofthe beta-fructofuranosidase enzyme; and c) separating thebeta-fructofuranosidase enzyme from the mycelium and from the culturemedium.
 2. Process, as in claim 1, characterized in that the culturemedium is defined by a semi-solid substrate, in such a way as to causethe production of the beta-fructofuranosidase enzyme in the free andexternal form in relation to the molecular structure of said fungus. 3.Process, as in claim 2, characterized in that the culture mediumcomprises a bran from a cereal containing starch, fibers and proteins,mineral salts and humidity.
 4. Process, as in claim 3, characterized inthat the culture medium comprises about 98% of cereal bran and about 2%of mineral fibers, which are adjusted to a humidity of about 30% to 60%in relation to the total weight of the culture medium.
 5. Process, as inclaim 4, characterized in that the cereal bran is wheat bran. 6.Process, as in claim 4, characterized in that the mineral salts includemagnesium sulfate, calcium chloride and potassium phosphate.
 7. Process,as in claim 1, characterized in that the fungus Aspergillus niger whichis cultivated is the one called 489, or a mutation or variation thereof.8. Process, as in claim 1, characterized in that the step of cultivatingthe fungus includes the incubation of said fungus at a temperature of30° C. for a period of 96 to 120 hours.
 9. A process for producingfructooligosaccharides, comprising sugars formed by one fructose unit,characterized in that it comprises the transfructosylation of thebeta-fructofuranosidase enzyme, which is prepared according to any ofthe claims 1-8.
 10. Process, as in claim 9, characterized in that thetransfructosylation of the enzyme is achieved in a solution with a pHranging from 5.0 to 9.0.